Cytotoxic T lymphocytes (CTLs) or "killer" T cells perform a crucial function in the vertebrate immune system by destroying cells that display foreign antigens on their surfaces. CTLs are distinguished from other types of T lymphocytes (such as "helper" T cells) by their expression of CD8, a transmembrane protein with immunoglobulin-like domains that interact with class I Major Histocompatibility Complexes (MHCs). MHC molecules have a dual role; their amino-terminal domains bind antigen for presentation to T cells and also serve as a marker identifying host cells to the immune system. Activation of CTLs to eliminate a target cell occurs only if the CD8 protein recognizes the proper MHC molecule and the α/β T cell receptor binds the MHC-presented antigen.

Historically, assays for an antigen-specific T cell response made use of the limiting dilution analysis (LDA) method, which requires that single clones survive, divide and differentiate before they can be detected. However, this method likely underestimates the number of CTLs that respond to a particular antigen because it will not detect cells that are part of the expanded effector population that can no longer divide. Alternate approaches attempt to directly measure CTL response using an antigenic peptide and MHC. However, these experiments are hampered by the low affinity of the interaction between the MHC/antigen complex and the corresponding CTL receptors.

Joining multiple copies of the MHC/antigen complex into a single probe—tetramer technology—resolves the difficulties presented by the low affinity of the class I MHC molecule for the CD8 receptor. CTL response assays using tetramer technology often detect a response rate that is 50–500 times higher than detected by the LDA method, and they have proven indispensable for the study of the CTL response to HIV, cancers and transplants. Similar methods using class II MHC tetramers are also being employed to explore the response of helper (CD4 expressing) T cells.

MHC tetramers are formed by first refolding MHCs in the presence of high concentrations of the desired antigenic peptide, followed by biotinylation of the carboxy-terminus of one chain of the MHC molecule. This MHC/peptide complex can then be bound to streptavidin. Because the latter has four biotin-binding sites, four MHC molecules can be linked together in a single complex (Figure 1). The use of fluorophore-labeled streptavidin or streptavidin-coated magnetic beads for tetramer formation allows for efficient detection by flow cytometry or immunomagnetic methods. It is critical for MHC tetramer–based assays that the labeled streptavidin be free of unlabeled streptavidin because this lowers the apparent activity of the MHC complex by "blocking" the biotinylation site from binding to the labeled streptavidin.